Materials and methods for controlling nematodes with pasteuria spores in seed coatings

ABSTRACT

The subject invention provides novel and advantageous materials and methods for controlling phytopathogenic and/or soil-dwelling nematodes by attaching an effective amount of  Pasteuria  spores to a seed and delivering the seeds to the situs of nematodes.

CROSS-REFERENCE TO A RELATED APPLICATION

The present application is a continuation application of co-pendingapplication Ser. No. 12/642,041, filed Dec. 18, 2009; which claims thebenefit of U.S. Provisional Application Ser. No. 61/139,304, filed Dec.19, 2008, all of which are hereby incorporated by reference in theirentirety, including all figures, tables or drawings.

BACKGROUND OF INVENTION

Crop losses due to plant parasitic nematodes are estimated to exceed$100 billion. Preventing this damage represents a significant challenge.With the impending loss of the fumigant methyl bromide, there isinsufficient time to develop and register new synthetic compounds fornematode control. Therefore, other options are needed.

Phytopathogenic nematodes are particularly difficult to control becausethey are covered with a thick, impermeable cuticle, or outer covering,and have very few sensory neurons. Since many pest control compoundsoperate as neurotoxins, the low number of neurons exposed byphytopathogenic nematodes decreases the effective target area fornematicidal compounds and has resulted in the development of nematicidalcompounds with extremely high neurotoxic properties.

Furthermore, because phytopathogenic nematodes are found in soil orplant roots, exposing the phytopathogenic nematodes to control agents isdifficult to achieve and puts the water table at risk of contaminationfrom these toxic compounds. The use of nematicides based on neurotoxinshas been demonstrated to contaminate both ground and surface water.Consequently, many of these compounds are being removed from the marketfor public health reasons.

Fumigation of soil prior to planting is a popular method for controllingnematodes. One of the most popular fumigants, methyl bromide, is slatedfor removal from use because of its ozone destroying properties.Furthermore, this practice of soil fumigation kills organisms in soilindiscriminately and runs the risk of eliminating beneficial microbes aswell as disease-causing organisms. Therefore, an effective nematicidewith benign environmental effects is urgently needed.

Pasteuria was first described in 1888 by Metchnikoff (Annales del'Institut Pasteur 2:165-170) as a parasite of water fleas.Subsequently, Cobb described a Pasteuria infection of the nematodeDorylaimus bulbiferous (2^(nd) ed. Hawaiian Sugar Planters Assoc., Expt.Sta. Div. Path. Physiol. Bull. 5:163-195, 1906).

The life cycle of the bacteria involves a stage when endospores bind tothe cuticle of the nematodes in soil. P. penetrans then proliferateswithin the nematode body and passes through several morphologicalphases, including mycelial structures and thalli, culminating in thedevelopment of new endospores. Endospores are released when the nematodebody lyses.

Growth of the bacteria within the nematode body reduces or eliminatesthe production of eggs by the nematode, severely restricting the rate ofnematode reproduction. Economic damage to the host crop normally isinflicted by the first generation progeny of nematodes and is preventedby Pasteuria through lowering the concentration of progeny nematodes inthe plant root zone.

Although the use of Pasteuria to control nematodes has been previouslyproposed, a number of factors, including sub-optimal delivery optionshave limited the use of this nematode control strategy. Conventionalmethods for controlling nematodes using Pasteuria strains includeapplying the bacteria to the plant and soil in free form (e.g. StirlingG. R. 1984. “Biological control of Meloidogyne javanica with Bacilluspenetrans”, Phytopathology, 74:55-60) or in solid and liquid formulation(e.g. U.S. Pat. No. 5,248,500). However, despite the highly selectiveeffectiveness of the bacteria on the phytopathogenic nematodes, theyneed to be in contact with the nematodes in order to produce thenematocidal effect. When applied directly to the soil, a large amount ofthe bacteria is required and they have to be mixed well with the soil,which substantially increases the cost of using the bacteria.

While various biocontrol methods using Pasteuria bacteria are known,there still remains a need for an improved approach for using thesebacteria to effectively control nematodes. Therefore, the subjectinvention provides novel methods for controlling phytopathogenicnematodes that attack plants.

BRIEF SUMMARY OF THE INVENTION

The subject invention provides novel and advantageous materials andmethods for controlling phytopathogenic nematodes and/or othersoil-dwelling nematodes by delivering to the soil a compositioncomprising an effective amount of Pasteuria spores that are attached toa seed.

Upon planting the seeds of the subject invention, the Pasteuria sporesare transferred to the soil surrounding the seed. It has been foundthat, when the method of the subject invention is practiced, the sporesthen attach to, infect, grow in, re-sporulate in, reduce the fecundityof and/or kill phytopathogenic nematodes and/or other soil-dwellingnematodes in the vicinity of the seed and, ultimately, any plant thatdevelops from the seed.

The method of the subject invention can be used for controlling orreducing harm caused by nematode infection, and, in preferredembodiments, can enhance seed emergence, plant growth and plant health.

One aspect of the present invention provides a method for nematodecontrol by delivering to the situs of a nematode infection an effectiveamount of Pasteuria spores attached to plant seeds.

The Pasteuria spores of the present invention can be applied to seeds asunformulated spores or as a formulated liquid or solid composition,slurry of particles, or emulsion. In one embodiment, Pasteuria sporesare formulated into a liquid composition. In another embodiment,Pasteuria spores are formulated into a solid composition. Suitable solidcarriers include but are not limited to solid polymeric matrices,particles, granules and powders. In one embodiment, the solid carrier ismade up of granules.

In preferred embodiments, the Pasteuria composition is associated withthe seeds by coating, spraying, or otherwise attaching to, contacting,or mixing the seeds with a Pasteuria composition. In one embodiment, thePasteuria composition is applied by coating at least part of the surfacearea of the seed with the Pasteuria composition.

In one embodiment, the plant seed is first treated with an adherent thatcan adhere to the Pasteuria spores and/or a composition containing thespores. The adherent can be, for example, a glue and/or one or morepolymers or copolymers. Examples of adherents include, but are notlimited to, glues (such as ELMERS™ glue); polyvinyl acetates; siliconematerials; and natural inorganic materials such as silica gel and clay.

Another aspect of the subject invention provides a seed having at leastpart of its surface coated with a Pasteuria composition, wherein thePasteuria composition comprises an effective amount of Pasteuria sporesfor nematode control.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the health of plants grown from seeds coated with Pasteuriafor nematode control.

FIG. 2 shows the attachment of Pasteuria spores to nematodes in thesoil.

FIG. 3 shows the height of plants grown from seeds coated with Pasteuriafor nematode control.

DETAILED DISCLOSURE

The present invention provides materials and methods for efficientcontrol of phytopathogenic nematodes and/or other soil-dwellingnematodes by utilizing plant seeds coated with Pasteuria spores.

Advantageously, Pasteuria produce endospores that have the unique anduseful property of being able to attach to, infect, grow in,re-sporulate in, reduce the fecundity of, and/or kill phytopathogenicnematodes and other soil-dewelling nematodes.

One aspect of the present invention provides methods for nematodecontrol by delivering, to the situs of a nematode infection, aneffective amount of a Pasteuria composition as a plant seed coating.

Pasteuria delivered as seed coatings according to the subject inventioncan reduce the ability of nematodes to infect plants. As a result, inpreferred embodiments, the subject treatment method is capable ofcontrolling or reducing harm caused by nematode infection, therebyenhancing seed emergence, plant growth and/or plant health.

Pasteuria spores of the present invention can be produced in vivo withinliving hosts (e.g. Verdeho, S., and R. Mankau, 1986, Journal ofNematology, 18:635), or in vitro without the presence of living hosttissues (e.g. U.S. Pat. No. 5,094,954). In a specific embodiment,Pasteuria spores of the present invention are produced usingfermentation techniques described in, for example, U.S. Pat. Nos.7,067,299 and 6,919,197.

Various Pasteuria species (Pasteuria spp.) are suitable for use fornematode control. In one embodiment, the active ingredient comprises aneffective amount of Pasteuria penetrans spores for controlling root-knotnematodes. In other embodiments, the active ingredient comprises aneffective amount of Pasteuria spores selected from Pasteuria ramose,Pasteuria thornea, Pasteuria usage, Pasteuria nishizawae, anycombination thereof, as well as new nematicidally species that will beidentified.

The nematicidally-effective amount of Pasteuria spores will vary,depending upon factors including, but not limited to, the plant species,the surface area of the seed, the type of carrier, presence or absenceof other active ingredients, the method of formulation, the route ofdelivery, the Pasteuria species, the target nematode species, and theseriousness of the nematode infection or damage to the plant(s).

“A nematicidally effective amount” as used herein refers to an amount ofPasteuria spores capable of killing, controlling, or infectingnematodes; retarding the growth or reproduction of nematodes; reducing anematode population; and/or reducing damage to plants caused bynematodes. In general, the effective amount of spores range from about1×10⁵ to 1×10¹² (or more) spores/seeds. Preferably, the sporeconcentration is about 1×10⁶ to about 1×10⁹ spores/seed.

An “active ingredient” as used herein refers to a substance that isuseful for killing, controlling, or infecting nematodes or other pests;and/or retarding the growth or reproduction of nematodes or other pests;reducing nematode or pest populations, and/or reducing damage to plantscaused by nematodes or other pest(s).

An “agriculturally beneficial ingredient” as used herein refers to asubstance that is useful or productive in agriculture settings, forexample, useful for controlling a disease, a pest (including for examplean insect, a parasite, a virus, a fungus, a bacterium) and/or a weed;useful for promoting the quantity and quality of plant growth, planthealth, seed emergence, plant reproduction and/or fruit growth.Agriculturally beneficial ingredients include but are not limited topesticides, herbicides, fungicides, fertilizers, and bio-control agents.

An “inert or inactive ingredient” as used herein refers to a substancethat aids in the operation or improves the effectiveness of anagricultural formulation or composition. Inert or inactive ingredientsinclude but are not limited to a carrier, an adherent, a dispersant, asurfactant, a liquid dilutant, a binder, a filler agent, a solvent, awetting agent, a sticker, an emulsifier, a nutrient, a surfactant, apenetrant, a foaming agent, a solubilizer, a spreader, and a bufferagent.

In certain embodiments, the Pasteuria composition is applied to theseeds by coating, spraying, attaching to, contacting, or mixing theseeds with the Pasteuria composition. In one embodiment, the Pasteuriacomposition is attached to the seed by coating at least part of thesurface area of the seed with the Pasteuria composition.

In a specific embodiment, the Pasteuria composition is applied to theseed by a) impregnating a solid carrier with the Pasteuria compositionto obtain a Pasteuria-carrier mixture; and b) contacting the seed withthe Pasteuria-carrier mixture.

Another aspect of the invention provides seeds treated with the subjectPasteuria composition. One embodiment provides seeds having at leastpart of the surface area coated with the Pasteuria composition. In aspecific embodiment, the Pasteuria treated seeds have a sporeconcentration from about 10⁶ to about 10⁹ spores per seed. The seeds mayalso have more spores per seed, such as, for example 1×10¹⁰, 1×10¹¹ or1×10¹² spores per seed.

Formulation of Pasteuria Composition

The Pasteuria of the present invention can be delivered to seeds asunformulated spores or as a formulated liquid or solid composition,slurry of particles, or emulsion.

In one embodiment, the Pasteuria spores are formulated into a liquidcomposition. Endospores are suspended in a medium buffered to maintainthe desired pH. Preferably, the pH is less than about 6.0, morepreferably the pH is less than about 5.5 and, most preferably, the pH isbetween about 3.0 and about 5.0. Buffer systems that can be usedinclude, but are not limited to, potassium hydrogen phthalate, aceticacid, succinic acid and citric acid. Agents that can be used to acidifythe culture medium include, but are not limited to, hydrochloric acid,sulfuric acid, acetic acid, and other organic acids.

In another embodiment, the Pasteuria composition may optionally compriseone or more amino acids, salts, carbohydrates, vitamins, and othersupporting nutrients. In a specific embodiment, the Pasteuria suspensionmay comprise one or more of the following components: glucose, NaCl,yeast extract, NH₄H₂PO₄, (NH₄)₂SO₄, glycerol, valine, L-leucine,L-glutamine, L-alanine, L-valine, L-tyrosine, L-tryptophan, lactic acid,propionic acid, succinic acid, malic acid, citric acid, KH tartrate,vitamin solution, mineral solution, xylose, lyxose, and lecithin. Inanother specific embodiment, the suspension may comprise one or more ofthe following components: lactic acid, propionic acid, succinic acid,malic acid, citric acid, tartaric acid, and yeast extract.

In one embodiment, Pasteuria spores are formulated into a solidcomposition. The solid suspension can be prepared by mixing Pasteuriaspores or a liquid Pasteuria suspension with a solid carrier. In oneembodiment, the solid composition is obtained by impregnating the solidcarrier with the spore suspension, and subsequently drying the solidcomposition.

Suitable solid carriers include, but are not limited to, solid polymericmatrices, particles, granules and powders. In one embodiment, the solidcarrier is made up of granules. In one embodiment, the subjectcomposition is formulated as an emulsion; slurry of particles in anaqueous medium (e.g., water); wettable powders; wettable granules (dryflowable); or dry granules. In a specific embodiment, the solid carrieris diatomaceous earth granules from AXIS® and/or greensgrade claygranules from PROFILE®.

The subject Pasteuria composition can be formulated as, for example, aliquid suspension, a solid composition, or aqueous slurry. Theconcentration of the active ingredient can range from about 0.5% toabout 99% by weight (w/w), about 5% to about 80%, about 10% to about75%, about 15% to about 70%, about 20% to about 65%, about 25% to about60%, about 30% to about 55%, about 35% to about 50%.

In another embodiment, the subject composition is formulated as aPasteuria-granule mixture. The amount of Pasteuria spores to granulescan range from about 1×10⁶ to about 7×10⁸ spores/g granules, about 5×10⁶to about 5×10⁸ spores/g granules, about 1×10⁷ to about 1×10⁸ spores/ggranules, or about 3×10⁷ to about 5×10⁷ spores/g granules.

In a specific embodiment, the granule composition is obtained by mixingabout 3-5 ml of a 2×10⁷ spores/ml Pasteuria spore suspension with about2 g granules. In a further specific embodiment, the granule compositionis obtained by mixing about 5 ml of the spore suspension with about 2 gAXIS® granules. In another further specific embodiment, the granulecomposition is obtained by mixing about 3 ml of the spore suspensionwith about 2 g PROFILE® granules.

Particles of the solid composition can be of any size capable ofattaching Pasteuria spores to a plant seed.

In a further embodiment, the Pasteuria composition further comprises oneor more conventional inactive or inert ingredients, including, forexample, adherents, dispersants, surfactants, liquid dilutants, binders,filler agents, solvents, wetting agents, stickers, emulsifiers,nutrients and buffer agents.

Conventional inactive or inert ingredients include, but are not limitedto: conventional sticking agents; dispersing agents such asmethylcellulose (METHOCEL™ A15LV or METHOCEL™ A15C, for example, serveas combined dispersant/sticking agents for use in seed treatments);polyvinyl alcohol (e.g., ELVANOL™ 51-05); lecithin (e.g., YELKINOL™ P),polymeric dispersants (e.g., polyvinylpyrrolidone/vinyl acetate PVPIVAS-630); thickeners (e.g., clay thickeners such as Van Gel B to improveviscosity and reduce settling of particle suspensions); emulsionstabilizers; surfactants; antifreeze compounds (e.g., urea), dyes,colorants, and the like. Additional inert ingredients useful in thepresent invention can be found in McCutcheon's, vol. 1, “Emulsifiers andDetergents,” MC Publishing Company, Glen Rock, N.J., U.S.A., 1996.Additional inert ingredients useful in the present invention can befound in McCutcheon's, vol. 2, “Functional Materials,” MC PublishingCompany, Glen Rock, N.J., U.S.A., 1996.

In one embodiment, an adherent is used to facilitate attachment of thespores to the seeds. The adherent attaches the spores or thePasteuria-containing composition to the surface of a seed, thuspreventing or at least reducing unwanted spore drop-offs. Preferably,the adherent is non-toxic, biodegradable, and adhesive. Suitableadherents include, but are not limited to glues; polyvinyl acetates;polyvinyl acetate copolymers; polyvinyl alcohols; polyvinyl alcoholcopolymers; celluloses, such as methyl celluloses, hydroxymethylcelluloses, and hydroxymethyl propyl celluloses; dextrins; alginates;sugars; molasses; polyvinyl pyrrolidones; polysaccharides; proteins;fats; oils; gum arabics; gelatins; syrups; and starches. Additionalsuitable adherents are described in, for example, U.S. Pat. No.7,213,367. In a specific embodiment, the adherent is polyvinyl acetate.

In another embodiment, the subject composition further comprises one ormore polymers capable of mixing or attaching the active ingredient to asolid carrier. Suitable polymers may be natural or synthetic, andpreferably have no or little phytotoxic effect on the seed to be coated.The polymer may be selected from, for example, polyvinyl acetates;polyvinyl acetate copolymers; ethylene vinyl acetate (EVA) copolymers;polyvinyl alcohols; polyvinyl alcohol copolymers; celluloses, includingethylcelluloses, methylcelluloses, hydroxymethylcelluloses,hydroxypropylcelluloses and carboxymethylcellulose;polyvinylpyrolidones; polysaccharides, including starch, modifiedstarch, dextrins, maltodextrins, alginate and chitosans; fats; oils;proteins, including gelatin and zeins; gum arabics; shellacs; vinylidenechloride and vinylidene chloride copolymers; calcium lignosulfonates;acrylic copolymers; polyvinylacrylates; polyethylene oxide; acrylamidepolymers and copolymers; polyhydroxyethyl acrylate, methylacrylamidemonomers; and polychloroprene.

In another embodiment, the subject composition may further comprise afiller agent for protecting the seeds during stress conditions. Inaddition, the subject composition may further comprise a plasticizer toimprove the fluidity of the liquid or semi-liquid composition, theflexibility of the mix or the polymeric composition, and/or theadhesiveness of the composition to the seed. In addition, it may bedesirable to add to the formulation a drying agent such as calciumcarbonate, kaolin or bentonite clay, perlite, diatomaceous earth or anyother adsorbent material as described for example in U.S. Pat. No.5,876,739. The skilled artisan, having the benefit of the currentdisclosure, can readily select desirable components to use in theformulation.

In yet a further embodiment, the subject Pasteuria composition comprisesa second agriculturally beneficial ingredient. The second agriculturallybeneficial ingredient could be, for example, selected from pesticidesand fungicides, such as captan, thiram, metalaxyl, fludioxonil,oxadixyl, and isomers of each of those materials, and the like;herbicides, including compounds such as carbamates, thiocarbamates,acetamides, triazines, dinitroanilines, glycerol ethers, pyridazinones,uracils, phenoxys, ureas, and benzoic acids; herbicidal safeners such asbenzoxazine, benzhydryl derivatives, N,N-diallyl dichloroacetamide,various dihaloacyl, oxazolidinyl and thiazolidinyl compounds, ethanone,naphthalic anhydride compounds, and oxime derivatives.

The second agriculturally beneficial ingredient may further comprisefertilizers and/or ingredients that promote seed germination, and/orplant growth and/or health. In addition, it may comprise variousbio-control agents such as other naturally-occurring or recombinantbacteria and fungi from the genera Rhizobium, Bacillus, Pseudomonas,Serratia, Trichoderma, Glomus, Gliocladium and mycorrhizal fungi. Thesechemical or biological agents may be beneficial for controlling nematodeand/or other pests.

In light of the above teachings, one skilled in the art would includevarious ingredients such as inert or inactive ingredients, pesticides,or fertilizers into the subject composition and/or treatment method. Inaddition, one skilled in the art would typically not include thoseingredients that would significantly harm the survival, growth, and/orreproduction of Pasteuria spores, reduce the ability of Pasteuria sporesto attach to, infect, grow in, and/or kill nematodes, and/or inhibit thegermination of seeds, plant growth, fruit growth and/or plantreproduction.

Plant Species

The materials and methods of the subject invention can be used forreducing damage to plant species, including, but not limited to, greenbeans, turf grasses, tomatoes, cotton, corn, soy beans, vegetables,wheat, barley, rice and canola.

Nematode Species

The materials and methods of the subject invention are useful forkilling, controlling, and/or infecting nematodes; retarding the growthor reproduction of nematodes; reducing nematode population; and/orreducing or retarding damage to plants caused by phytopathogenicnematodes, plant-parasitic nematodes, and other soil-dwelling nematodes,including but not limited to Meloidogyne arenaria, Pratylenchusbrachyurus, Rotylenchulus reniformis, and Belonolaimus longicaudatus,Heterodera glycines and Hoplolaimus galeatus.

Methods of Attachment and Delivery

The present invention also provides methods for attaching the Pasteuriacomposition to plant seeds and delivering the seeds to a situs ofnematode infection.

It is preferable that the attachment method does not significantly harmthe survival, growth, and/or reproduction of Pasteuria spores, reducethe ability of Pasteuria spores to attach to, infect, grow in, and/orkill nematodes. Preferably, the attachment and delivery methods wouldproduce little phytotoxicity, such as affecting seed germination, plantvascularization, plant height, plant reproduction and/or fruit growth.

In certain embodiments, the Pasteuria composition is attached bycoating, spraying, contacting, or mixing the seed with the Pasteuriacomposition. In one embodiment, the Pasteuria composition is attached bycoating at least part of the surface area of the seed with the Pasteuriacomposition.

The subject composition can be attached to seeds using any of a varietyof techniques, such as, for example, fluidized bed techniques, theroller mill methods, and spouted bed techniques. In addition, thesubject composition can be attached to seeds using a machine, such as arotostatic seed treater or a drum coater. The seeds may be pre-sizedbefore coating. After coating, the seeds are typically dried and thentransferred to a sizing machine for sizing, as is known in the art.

In one embodiment, the subject Pasteuria composition is first mixed withvarious agriculturally beneficial ingredients such as pesticides,herbicides, fungicides, fertilizers and/or various bio-control agents;and the mixture is then delivered to the seeds. In another embodiment,the subject Pasteuria composition is delivered sequentially with variousagriculturally beneficial ingredients to the seeds. In anotherembodiment, the subject Pasteuria composition is deliveredsimultaneously with various agriculturally beneficial ingredients, butto different areas of the seed surface.

As used herein, seed coating includes any process that adds materials tothe seed, including but not limited to, film coating, deposition of asingle layer or layers of materials to the entire or part of the surfacearea of a seed, application of continuous layers of materials to theentire or part of the surface area of a seed, application of materialsto the seed simultaneously or sequentially to cover the entire or partof the surface area of a seed.

In specific embodiments, the subject formulation can be coated on seedsusing a variety of methods, including but not limited to, film coating,mixing seeds with the subject formulation in a container (e.g., a bottleor bag), mechanical application, tumbling, spraying, and immersion. Avariety of active or inert materials can be used for coating, such asfor example conventional film-coating materials including but notlimited to water-based film coating materials such as SEPIRET (Seppic,Inc., Fairfield, N.J.) and Opacoat (Berwind Pharm. Services, Westpoint,Pa.).

Various methods for producing coated seeds further include thosedescribed in for example, U.S. Pat. Nos. 5,918,413; 5,891,246;5,554,445; 5,389,399; 5,107,787; 5,080,925; 4,759,945; 4,465,017;5,939,356; 5,882,713; 5,876,739; 5,849,320; 5,834,447; 5,791,084;5,661,103; 5,622,003; 5,580,544; 5,328,942; 5,300,127; 4,735,015;4,634,587; 4,383,391; 4,372,080; 4,339,456; 4,272,417; and 4,245,432.

In another specific embodiment, the Pasteuria composition is deliveredto seeds by solid matrix priming. To briefly illustrate, Pasteuriaspores uniformly distributed in a solid matrix are placed in contactwith seeds for a sufficient amount of time until the entire surface areaof the seeds is covered with Pasteuria spores. The treated seeds can beseparated from the solid matrix for further storage or use; or,alternatively, the treated seeds can be stored or planted together withthe solid matrix.

Materials suitable for use as a solid matrix include polyacrylamide,starch, clay, silica, alumina, soil, sand, polyurea, polyacrylate, orany material capable of absorbing and releasing the subject compositiononto seeds in a controlled manner. Preferably, the solid matrix iscapable of releasing the subject composition in a controlled manner. Thedesired release rate may vary depending on the plant species, thePasteuria species, the nematode species and other factors. Preferably,the subject composition can be released from the treated seeds at slowrate, such as by diffusion or moving through the matrix to thesurrounding medium or the soil.

Seeds coated with the Pasteuria composition can be further envelopedwith an additional thin film of over-coating to protect the Pasteuriacoating. Exemplified techniques for over-coating include, but are notlimited to, the fluidized bed technique and the drum film coatingtechnique.

In a further embodiment, the delivery methods include an additionalheat-treatment step. The heat-treatment step would enhance thenematicidal effect of the Pasteuria composition. In one embodiment, thePasteuria spore suspension is heated before mixing with a solid carriersuch as granules. In another embodiment, the Pasteuria-granule mixtureis heated.

In addition, the subject method can comprise a step of applying anadherent to the seeds prior to or simultaneously with the Pasteuriatreatment. In one embodiment, the seeds are first coated with a layer ofadherent, and then treated with the Pasteuria composition. Suitableadherents include but are not limited to polyvinyl acetate, polyvinylacetate copolymers, polyvinyl alcohol, polyvinyl alcohol copolymer,methyl cellulose, hydroxymethyl cellulose, hydroxymethyl propylcellulose dextrin, alginate, molasse, polyvinyl pyrrolidone,polysaccharides, protein, fat, oil, polysaccharide, gum Arabic, gelatin,syrups, and any of the combination thereof.

In a specific embodiment, the subject method comprises a) mixing aPasteuria spore suspension with a particulate carrier, and drying themixture for a sufficient time until spore/granule particles are formed;b) coating seeds with an adherent; and c) contacting the seeds with thespore/granule particles until the seed is coated with anematicidally-effective amount of the Pasteuria spores.

In yet a further embodiment, seeds treated with the subject Pasteuriacomposition can undergo additional processing steps such as drying.Advantageously, Pasteuria spores would not be damaged by drying. Thus,seeds treated with the subject composition can be stored for a prolongedperiod of time at room temperature. The long shelf life of the treatedseeds also allows for variations in planting schedules. Advantageously,the survival rate of the Pasteuria spores is much higher than thesurvival rate of the vegetative form of the bacteria during transport,sowing, or once placed in the soil with seeds.

Although the seed treatments can be applied to a seed in anyphysiological state, it is preferred that the seed is in a sufficientlydurable state so that the treatment process will produce little or nodamage to the seed. Typically, the plant has been harvested from thefield; the seed removed from the plant; and separated from any non-seedplant material. The seed is preferably biologically stable to the extentthat the treatment does not cause damage to the seed. In one embodiment,for example, the treatment can be applied to seed corn that has beenharvested, cleaned and dried to moisture content below about 15% byweight.

In an alternative embodiment, the seed can be one that has been driedand then primed with water and/or other materials and then re-driedbefore or during the treatment with the Pasteuria composition. Thetreatment can be applied to seeds at any time from harvest to sowing. Asused herein, the term “unsown seed” includes any seed at any period fromharvest to sowing.

Following are examples, which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Example 1 Preparation of Pasteuria Seed Coating

This Example illustrates methods for coating seeds with Pasteuriapenetrans spores. Pasteuria spores are suspended in 10 mmol/L phosphatebuffer and adjusted to about 2×10⁷ spores/ml. A Pasteuria-granularmixture is obtained by mixing 5 ml of the spore suspension with 2 gAXIS® diatomaceous earth granules in a petri dish; or alternatively, bymixing 3 ml spore suspension with 2 g PROFILE® greens grade claygranules in a petri dish.

The mixture is dried under a lamp. Then, green bean seeds are treatedwith polyvinyl acetate and allowed to dry for 5 minutes in a flat dish.The seeds are thoroughly coated with the Pasteuria-granular mixture.

The Pasteuria coated seeds are suitable for immediate use or long termstorage.

Example 2 Efficacy of Pasteuria-Coating of Seeds for Nematode Control

To evaluate the effect of Pasteuria-coated seeds on nematodes, agreenhouse pot test is performed.

2.2 g each of Axis turf supplement and Profile turf supplement wereplaced in separate petri dishes. Pasteuria penetrans spores at aconcentration of 1.8×10⁷ sp/ml were pipetted into each dish until thematerials were saturated. The Axis supplement was saturated at 5 mL andthe Profile was saturated at 4 mL. The dishes were set under a halogenlamp to dry. Green bean seeds (Ferry-Morse Blue Lake 274) were weighedand determined to have an average weight of 413.3 mg. Seeds were coatedwith a polyvinyl acetate sticker by pouring Elmer's Clear School Glueinto a beaker and manually dunking individual seeds with tweezers intothe glue. Seeds were dried for 5 minutes in a petri dish, then rolled inthe spore-treated Axis and Profile granules until thoroughly coated.Control seeds were coated as above with untreated Axis and Profilegranules.

The coated seeds are exposed, as follows, to field-collected root-knotnematodes for 21 days. In the control, non-treated green bean seeds areexposed to nematodes under the same conditions.

Styrofoam cups were filled with 1 kg of soil. Each pot was inoculatedwith 1500-2000 root-knot juveniles (Meloidogyne arenaria) by pipetting asuspension of the nematodes into the soil at a depth of 2 inches belowthe soil surface. The resulting hole was filled, then the green beanseeds were planted, one per pot, at a depth of 0.5 inches below thesurface. 5 test (with spores) and 5 control seeds were planted. Potswere watered with 50 ml tap water daily, and maintained in thegreenhouse until temperature readings indicated the nematodes hadreached 500 degree-days.

Soil was then sampled for nematode counts, and plants and root systemsremoved from soil. Plant health and vigor was evaluated on a scale of1-10, 1 being the least healthy and 10 the most healthy. Percentattachment was evaluated by recording the number of juvenile nematodesin a soil sample with 1 or more Pasteuria spores attached andcalculating the percentage. Nematode eggs were counted by collecting eggmasses from the root systems and counting the eggs microscopically.

The results, shown in FIGS. 1 through 3, demonstrate that Pasteuriacoating of seeds promotes seed emergence, plant height, and plant growthcompared to the control treatment.

All references, including patents, patent applications, provisionalapplications, and publications referred to or cited herein areincorporated by reference in their entirety, including all figures andtables, to the extent they are not inconsistent with the explicitteachings of this specification.

The terms “a” and “an” and “the” and similar referents as used in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise indicated. No language in the specification should beconstrued as indicating any element is essential to the practice of theinvention unless as much is explicitly stated.

The description herein of any aspect or embodiment of the inventionusing terms such as “comprising”, “having”, “including” or “containing”with reference to an element or elements is intended to provide supportfor a similar aspect or embodiment of the invention that “consists of”,“consists essentially of”, or “substantially comprises” that particularelement or elements, unless otherwise stated or clearly contradicted bycontext (e.g., a composition described herein as comprising a particularelement should be understood as also describing a composition consistingof that element, unless otherwise stated or clearly contradicted bycontext).

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

We claim:
 1. A method for controlling phytopathogenic and/orsoil-dwelling nematodes, comprising attaching a Pasteuria compositioncomprising an effective amount of Pasteuria spores to a seed; andplanting the seed.
 2. The method, according to claim 1, wherein thePasteuria composition, when applied to the seed, is present at aconcentration ranging from about 10⁶ to about 10⁹ spores per seed. 3.The method, according to claim 1, wherein the Pasteuria compositionfurther comprises a liquid or solid carrier.
 4. The method, according toclaim 3, wherein the solid carrier is selected from the group consistingof dry powders, wettable powders, wettable granules, dry granules,particles, polymeric matrices, and slurries.
 5. The method, according toclaim 4, wherein the solid carrier comprises granules.
 6. The method,according to claim 4, wherein the solid carrier is a polymeric matrixselected from the group consisting of polyacrylamide, starch, clay,silica, alumina, soil, sand, polyurea, polyacrylate, and any of thecombination thereof.
 7. The method, according to claim 1, wherein anadherent has been applied to the seed, and wherein the adherent isselected from the group consisting of polyvinyl acetate, polyvinylacetate copolymers, polyvinyl alcohol, polyvinyl alcohol copolymer,methyl cellulose, hydroxymethyl cellulose, hydroxymethyl propylcellulose dextrin, alginate, molasses, polyvinyl pyrrolidone,polysaccharides, protein, fat, oil, polysaccharide, gum Arabic, gelatin,syrups, and any combination thereof.
 8. The method, according to claim1, wherein the Pasteuria spores are selected from the group consistingof Pasteuria penetrans, Pasteuria ramose, Pasteuria thornei, Pasteurianishizawae, and any combination thereof.
 9. The method, according toclaim 1, wherein the phytopathogenic and/or soil-dwelling nematodes areselected from the group consisting of Meloidogyne arenaria, Pratylenchusbrachyurus, Rotylenchulus reniformis, Belonolaimus longicaudatus, andany combination thereof.
 10. The method, according to claim 1, whereinthe seed is selected from the group consisting of seeds for green beans,tomatoes, cotton, corn, soy, wheat, barley, rice, canola, and anycombination thereof.
 11. The method, according to claim 1, wherein theseed further comprises a second agriculturally beneficial ingredientselected from the group consisting of pesticides, herbicides,fungicides, fertilizers, and bio-control agents.
 12. A seed having atleast part of the surface area coated with a Pasteuria composition,wherein the Pasteuria composition comprises an effective amount ofPasteuria spores for nematode control.